Abstract

In the 1920s, it was recognized that a mucoid or matte colony phenotype of Streptococcus pyogenes was associated with virulence in mice and with resistance to killing by human blood leukocytes. The gelatinous material produced by the mucoid isolates, currently known as capsule, was later characterized as hyaluronic acid, a linear polymer of N-acetylglucosamine and glucuronic acid with a high molecular mass that is structurally identical to hyaluronic acid found in the extracellular matrix of many higher animals, including human beings. It is now recognized that most (but not all) clinical isolates of group A streptococci produce hyaluronic acid, which is associated with the cell surface during exponential growth and shed during stationary phase. This chapter presents a summary of information on the capsule of Streptococcus pyogenes, its biochemistry, genetics, and role in virulence.

Introduction

For many years, clinical microbiologists and infectious diseases clinicians have noted that group A streptococci freshly isolated from patients with pharyngitis or invasive infection often grow as large, wet-appearing, translucent colonies on blood agar. With prolonged incubation, these “mucoid” colonies collapse and assume an irregular or “matte” appearance. Laboratory passage of such isolates frequently resulted in loss of the mucoid phenotype and conversion to small, compact, or “glossy” colonies (Figure 1). In the 1920s, Lancefield and Todd recognized that the mucoid or matte colony phenotype was associated with virulence in mice and with resistance to killing by human blood leukocytes (Lancefield & Todd, 1928; Todd & Lancefield, 1928). Kendall and co-workers characterized the gelatinous material produced by mucoid isolates as hyaluronic acid, a linear polymer of N-acetylglucosamine and glucuronic acid with a high molecular mass that is structurally identical to hyaluronic acid found in the extracellular matrix of many higher animals, including human beings (Figure 2) (Kendall, Heidelberger, & Dawson, 1937). Wilson demonstrated that the growth of such isolates on media containing hyaluronidase prevented the development of mucoidy, which confirmed that the production of hyaluronic acid was responsible for the mucoid colony phenotype (Wilson, 1959). It is now recognized that most (but not all) clinical isolates of group A streptococci produce hyaluronic acid, which is associated with the cell surface during exponential growth and shed during stationary phase. Presumably because it is recognized by the host immune system as a self-antigen, the group A streptococcal hyaluronic acid capsule is poorly immunogenic in animals, including human beings. In contrast to capsular polysaccharides of Streptococcus pneumoniae and S. agalactiae, hyaluronic acid polymers are not covalently linked to the group A streptococcal cell wall, but rather are associated with the cell surface in a dynamic fashion, as they are synthesized by a cell membrane-associated polymerase. Although its mode of attachment to the bacterial surface is more tenuous than that of covalently bound polysaccharide capsules in other species, there is abundant evidence that the hyaluronic acid capsule is an important virulence determinant as a modulator of multiple interactions between group A streptococci and their human hosts.

Figure 1.

Blood agar plate with typical colonies of a mucoid strain of group A streptococcus (left) and non-mucoid (glossy) colonies of an acapsular mutant (right).

Figure 2.

Repeating unit structure of hyaluronic acid.

Genetics and biochemistry of hyaluronic acid biosynthesis

Studies in the 1990s used transposon mutagenesis to identify a chromosomal locus required for hyaluronic acid production in group A streptococci (DeAngelis, Papaconstantinou, & Weigel, 1993a; Dougherty & van de Rijn, 1992; Wessels, Moses, Goldberg, & DiCesare, 1991). Further characterization of the locus revealed an operon of three genes, hasA, hasB, and hasC, each of which encodes an enzyme involved in hyaluronic acid synthesis (DeAngelis, Papaconstantinou, & Weigel, 1993a; Crater, Dougherty, & van de Rijn, 1995; DeAngelis, Papaconstantinou, & Weigel, 1993b; Dougherty & van de Rijn, 1993; Dougherty & van de Rijn, 1994) (Figure 3). The 4.2 kb has operon is highly conserved among group A streptococcal strains, but is absent in isolates of M-types 4 and 22, which do not produce capsules (Henningham, et al., 2014; Flores, Jewell, Fittipaldi, Beres, & Musser, 2012). Notably, strains of M-types 4 and 22 produce hyaluronidase, which degrades hyaluronic acid, while the vast majority of other group A streptococcal isolates harbor an inactivating point mutation in the chromosomal hyaluronidase gene, hylA (Henningham, et al., 2014; Hynes, Johnson, & Stokes, 2009). This association suggests an evolutionary branch point in which group A streptococcal strains developed alternative strategies for adaptation through expression of either the anti-phagocytic hyaluronic acid capsule or hyaluronidase, which facilitates the spread of secreted toxins by degrading the host’s extracellular matrix, but can also digest the group A streptococcus capsule.

Figure 3.

(A) Schematic of the has operon encoding enzymes involved in hyaluronic acid biosynthesis in group A streptococci. (B) Diagram illustrating the enzymatic function in hyaluronic acid synthesis of the proteins encoded by hasA, hasB, and hasC.

A new clade of emm89 group A streptococci was identified in the United Kingdom in 2008 as a rapidly expanding population among isolates from invasive infections. The isolates, like the invasive emm1 clonal group that emerged in the 1980s, appeared to have acquired a chromosomal segment that included polymorphisms responsible for high-level expression of streptolysin O and NAD-glycohydrolase (Turner, et al., 2015; Zhu, Olsen, Nasser, Morales, & Musser, 2015). In addition, and in contrast to the emm1 strains, the emergent emm89 clade harbored a complete deletion of the has operon. That the acapsular emm89 strains are clearly capable of causing invasive infections suggests that high level expression of streptolysin O and NAD-glycohydrolase and perhaps other virulence determinants may compensate for the loss of capsule with respect to pathogenesis. Group A streptococci of emm types 28 and 87 also do not produce capsule. These strains contain the has operon, but harbor a single nucleotide insertion in hasA that results in a frame shift, premature termination of translation, and a non-functional product (Flores, McNeil, Shah, Van Beneden, & Shelburne, 2019).

Hyaluronic acid is synthesized from the nucleotide sugar precursors UDP-glucuronic acid and UDP-N-acetylglucosamine by a membrane-associated enzyme, hyaluronan synthase, encoded by hasA. High-Mr hyaluronic acid can be produced by the incubation of cell-free membrane extracts of group A streptococci with the two substrate UDP-sugars in the presence of divalent cations (Markovitz, Cifonelli, & Dorfman, 1959; Stoolmiller & Dorfman, 1969; Sugahara, Schwartz, & Dorfman, 1979). The hasA gene product has a predicted Mr of 47.9 kD and includes at least four predicted membrane-spanning domains, which is consistent with evidence that the enzyme is localized at the cell membrane where it mediates both polymer formation and export (DeAngelis, Papaconstantinou, & Weigel, 1993a; Dougherty & van de Rijn, 1994). The group A streptococcus hyaluronan synthase shares significant similarity with hyaluronan synthases from other microbial and higher animal species (DeAngelis, Yang, & Weigel, 1994; Weigel, Hascall, & Tammi, 1997). The second gene of the has operon, hasB, encodes UDP-glucose dehydrogenase, a 45.5 kD protein that catalyzes the oxidation of UDP-glucose to UDP-glucuronic acid (Dougherty & van de Rijn, 1993). The third gene in the cluster, hasC, encodes a predicted 33.7 kD protein identified as UDP-glucose pyrophosphorylase (Crater, Dougherty, & van de Rijn, 1995). This enzyme catalyzes the condensation of UTP with glucose-1-phosphate to form UDP-glucose. Thus, the reaction catalyzed by the hasC product yields a substrate for UDP-glucose dehydrogenase encoded by hasB, whose reaction product is, in turn, a substrate for hyaluronan synthase encoded by hasA. While the enzyme protein encoded by hasC is enzymatically active, it is not required for hyaluronic acid synthesis by group A streptococci. The inactivation of hasC resulted in no reduction in hyaluronic acid synthesis by a highly encapsulated strain of group A streptococci—a finding that implies that another source of UDP-glucose is available within the cell (Ashbaugh, Alberti, & Wessels, 1998a). Furthermore, expression of recombinant hasA and hasB (without hasC) conferred the capacity to synthesize hyaluronic acid in Escherichia coli and Enterococcus faecalis (DeAngelis, Papaconstantinou, & Weigel, 1993a; DeAngelis, Papaconstantinou, & Weigel, 1993b). A hasB paralog, hasB2, is widely conserved among group A streptococcus isolates. This gene is located at a site remote from the has operon; it encodes a protein with the same enzymatic activity as hasB and can support a reduced level of capsule synthesis in the absence of hasB (Cole, Aziz, Kuipers, Timmer, Nizet, & van Sorge, 2012).

Regulation of capsule expression

While the has operon is highly conserved, there is wide variation in production of the hyaluronic acid capsule among group A streptococcus isolates and under different growth conditions in an individual strain. Transcription of the has operon and synthesis of hyaluronic acid is maximal during exponential phase in liquid cultures, and declines to very low levels during the stationary phase (Crater & van de Rijn, 1995; Unnikrishnan, Cohen, & Sriskandan, 1999). Cessation of capsule synthesis is associated with shedding of hyaluronic acid from the cell surface into the culture medium. Capsule production is highest in a nutrient-rich environment. Expression of the has operon is rapidly induced upon introduction of the bacteria into the peritoneal cavity of mice or into the pharynx of baboons in a non-human primate model of pharyngeal colonization (Gryllos, Cywes, Shearer, Cary, Kennedy, & Wessels, 2001). Strain-to-strain variation in capsule production may be partially explained by polymorphisms in the promoter region upstream of the has operon (Albertí, Ashbaugh, & Wessels, 1998).

The CsrRS (also known as CovRS) two-component system is a critical regulator of has operon transcription in response to environmental signals. The CsrRS system was discovered by a transposon mutagenesis screen for mutants that formed mucoid colonies; a phenotype that is shown to be the result of inactivation of the csrRS locus (Levin & Wessels, 1998). Subsequent work has shown that CsrRS regulates approximately 10% of group A streptococcal genes, including several virulence factors, in addition to the has operon (Dalton, Collins, Barnett, & Scott, 2006; Federle, McIver, & Scott, 1999; Graham, et al., 2002; Gryllos, et al., 2007; Heath, DiRita, Barg, & Engleberg, 1999). According to our current model of the CsrRS system, CsrS is a cell membrane-associated histidine kinase that is activated by extracellular magnesium, and perhaps also by other unknown environmental signals (Gryllos, et al., 2007; Gryllos, Levin, & Wessels, 2003). Autophosphorylation of the cytoplasmic domain of CsrS is followed by phosphotransfer to CsrR, which increases the affinity of the latter protein for target promoters—including the has operon promoter, where it represses transcription and reduces capsule production (Horstmann, et al., 2015). Chromatin immunoprecipitation and DNA sequencing analysis has shown that repression of has operon expression correlates with CsrR occupancy at the operon promoter (Finn, Ramsey, Dove, & Wessels, 2021). The human antimicrobial peptide LL-37 appears to signal through the CsrRS system in a manner opposite to that of magnesium: exposure of group A streptococci to 100-300 nM LL-37 (concentrations far below those that inhibit bacterial growth) results in reduced phosphorylation of CsrR, reduced occupancy at the has operon promoter, and derepressed expression of the has operon (and of other CsrR-repressed genes) (Gryllos, et al., 2008; Tran-Winkler, Love, Gryllos, & Wessels, 2011; Finn, Ramsey, Dove, & Wessels, 2021). LL-37 has been shown to bind to the predicted extracellular domain of CsrS in vitro, and studies of smaller peptides have identified a 10-amino acid internal fragment of LL-37 that is completely devoid of antimicrobial activity against group A streptococci, but retains its CsrS-signaling activity (Velarde, Ashbaugh, & Wessels, 2014). These findings suggest that LL-37 signaling reflects a specific interaction with CsrS rather than a response to a non-specific membrane injury. It has been suggested that the CsrRS system enables group A streptococci to detect the host’s innate immune response to group A streptococcal infection by sensing subinhibitory concentrations of LL-37 secreted by leukocytes and/or epithelial cells. LL-37 signaling through CsrS results in upregulation of capsule production, as well as that of other antiphagocytic factors that enhance group A streptococcal virulence (Gryllos, et al., 2008; Tran-Winkler, Love, Gryllos, & Wessels, 2011).

RocA is another regulatory protein that affects capsule expression. Inactivating mutations in rocA have been shown to reduce expression of CsrR and are associated with increased capsule production (Biswas & Scott, 2003). Subsequent studies have characterized RocA as an auxiliary regulator of the CsrRS system. The rocA sequence is mutated in both emm18 and emm3 strains so both emm types produce truncated versions of RocA that are defective in modulating regulation of CsrRS-regulated genes, resulting in increased expression of capsule and several other virulence factor (Lynskey, et al., 2013; Miller, et al., 2015). Replacement of the mutated rocA gene with the consensus rocA sequence in a mucoid M18 strain suppressed the mucoid phenotype (Lynskey, et al., 2013). Interaction of RocA with CsrS in the streptococcal cell membrane appears to be essential for RocA-mediated regulation of CsrRS function (Lynskey, Velarde, Finn, Dove, & Wessels, 2019).

Role of the capsule in pathogenesis

Early studies by Lancefield, Todd, and others found an association between the presence of the mucoid colony type and virulence in mice (Todd & Lancefield, 1928; Ward & Lyons, 1935). However, because mucoid strains tended to be rich in M protein as well as in hyaluronic acid capsule, it was difficult to confidently ascribe an independent role in virulence to the capsule. Investigators in the mid-twentieth century found that hyaluronidase treatment increased the susceptibility of mucoid strains to killing by human blood phagocytes, supporting a role for the capsule in resistance to phagocytosis (Rothbard, 1948; Foley & Wood, Jr., 1959; Stollerman, Rytel, & Ortiz, 1963). Kass and Seastone showed that hyaluronidase treatment of mice reduced the virulence of group A streptococci during experimental infections (Kass & Seastone, 1944). Epidemiological observations have also suggested a link between capsule expression and virulence. Mucoid strains of group A streptococci have been associated with invasive infections and with outbreaks of acute rheumatic fever (Johnson, Stevens, & Kaplan, 1992; Tamayo, Montes, García-Medina, García-Arenzana, & Pérez-Trallero, 2010; Veasy, et al., 2004).

The development of methods for genetic manipulation of streptococci permitted more direct assessment of the role of the capsule in pathogenesis. Acapsular mutants derived by transposon mutagenesis and subsequently by targeted deletion of the hasA gene were found to have reduced virulence in systemic infection models in mice and in chicken embryos, in a murine model of invasive soft tissue infection, and in airway challenge models in mice (Wessels, Moses, Goldberg, & DiCesare, 1991; Ashbaugh, Warren, Carey, & Wessels, 1998b; Husmann, Yung, Hollingshead, & Scott, 1997; Schmidt, Günther, & Courtney, 1996; Schrager, Rheinwald, & Wessels, 1996; Wessels & Bronze, 1994). Capsule-deficient mutants have increased susceptibility to complement-mediated opsonophagocytic killing by human blood leukocytes, as compared to their respective encapsulated parent strains, and resistance to phagocytosis is thought to be a major mechanism through which the capsule enhances virulence. The presence of the capsule does not inhibit complement activation or deposition of complement fragments on the bacterial cell wall, but rather interferes with access of leukocyte receptors for opsonic complement proteins on the bacterial surface (Dale, Washburn, Marques, & Wessels, 1996).

In a baboon model of group A streptococcal pharyngeal colonization, an acapsular mutant colonized at a similar efficiency as the parent strain, but was cleared more rapidly (Ashbaugh, Moser, Shearer, White, Kennedy, & Wessels, 2000). This result suggested that the capsule contributed to persistence in the pharynx. However, genomic analysis of serial isolates from the pharynges of experimentally infected macaques showed the development over time of mutations in the has operon and promoter that reduced transcription of the hyaluronic acid biosynthesis genes (Shea, et al., 2011). Similar mutations were detected in sequential group A streptococcal isolates from human pharyngeal samples (Flores, et al., 2014). When taken together, these observations suggest that the down-regulation of capsule production may favor chronic pharyngeal carriage.

In vitro studies of group A streptococcal adherence to epithelial cells have shown that the capsule reduces bacterial attachment (Hollands, et al., 2010; Schrager, Albertí, Cywes, Dougherty, & Wessels, 1998). The capsule itself can act as an adhesin by mediating attachment to the hyaluronic acid binding protein CD44, which is expressed on multiple cell types including oropharyngeal keratinocytes (Schrager, Albertí, Cywes, Dougherty, & Wessels, 1998). The potential role of CD44 as a group A streptococcus receptor was supported by studies showing reduced pharyngeal colonization in mice after intranasal administration of monoclonal antibody to CD44 together with a group A streptococcal challenge or after pretreatment with exogenous hyaluronic acid (Cywes, Stamenkovic, & Wessels, 2000). Mice expressing a CD44 anti-sense transgene targeted to stratified squamous epithelia also were resistant to group A streptococcal pharyngeal colonization. In addition to the role of CD44 in adherence, the binding of encapsulated group A streptococcus to CD44 on human oropharyngeal keratinocytes induces an intracellular signaling cascade that results in disruption of intercellular junctions and enhancement of group A streptococcal translocation across the epithelial barrier. In this way, CD44-mediated signaling by the hyaluronic acid capsule may facilitate group A streptococcus tissue invasion (Cywes & Wessels, 2001).

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